Carbon dioxide in blood serum or blood plasma is in an equilibrium with bicarbonate ion (HCO.sub.3.sup.-) and is a second largest fraction in blood serum and blood plasma. Therefore, the carbon dioxide forms the most important biological buffering action system in blood. The carbon dioxide content in blood serum or blood plasma is a significant index of electrolytic dispersion and shortage of anion, to aid medical diagnosis of acid-base inequilibrium in the respiratory system and metabolism. For example, normal bicarbonate ion concentration in blood serum and blood plasma is 22-32 mmol/l and a decrease to a low value of 15 mmol/l or an increase to a high value of 40 mmol/l suggests presence of abnormality.
While a carbon dioxide level in body fluids can be measured by various methods, it is generally measured by tracing the changes caused by the enzyme reaction to be mentioned later. When phosphoenolpyruvate carboxylase is reacted with bicarbonate ion and phosphoenolpyruvic acid, oxalacetic acid and phosphoric acid are produced. The resultant oxalacetic acid is reacted with malate dehydrogenase in the presence of NADH, and a decrease in NADH is measured by a known method such as an end point method or a rate method.
In the alkaline pH range, the equilibrium between carbon dioxide and bicarbonate ion shifts toward the reaction to form bicarbonate ion, and carbon dioxide exists as bicarbonate ion; in the acidic pH range, it forms CO.sub.2 gas which is released into the air. For this reason, buffers having pH of 8.0 or above are generally used for a reagent for determining carbon dioxide.
The basic problem associated therewith is that phosphoenolpyruvate carboxylase is unstable in the alkaline pH range, with the result that a reagent for carbon dioxide measurement containing this enzyme cannot be stored for a long time in a liquid state. The phosphoenolpyruvate carboxylases available in the market are mostly derived from plants such as maize leaves and wheat germ, and show extremely poor stability at pH 8.0.
There is also known measurement of bicarbonate ion by the use of phosphoenolpyruvate carboxylase derived from Hyphomicrobium (EP 456444). This enzyme has a microbial origin, is free of the above-mentioned defects that the plant-originated phosphoenolpyruvate carboxylase has, and shows relatively high stability. It has been found, however, that this enzyme is not sufficiently stable at the enzyme concentration (generally 0.05-5 U/ml) that a reagent for measuring carbon dioxide generally has.
In the measurement method as disclosed in U.S. Pat. No. 3,974,037, phosphoenolpyruvate carboxylase derived from Escherichia coli is used. This enzyme shows poor stability in a reagent solution for measuring carbon dioxide which is weak alkaline.
Accordingly, a phosphoenolpyruvate carboxylase which is stable in a buffer (having a pH of about 8.0-8.5) to be contained in the CO.sub.2 measurement reagent is demanded.